Genetically attenuated bacterial vaccines with multiple mutations of the same phenotype

ABSTRACT

The present invention relates to a vaccine composition for the prevention of infectious diseases comprising an immunogenically-effective amount of a genetically attenuated and stable avirulent bacterial strain derived from a virulent bacterial strain by introducing into said virulent strain by genetic transfer and recombination at least two mutations of the same phenotype which renders said virulent strain avirulent, while permitting said avirulent strain to retain immunogenicity, together with an inert pharmacologically acceptable carrier. It also relates to a method for preparing the vaccine.

This is a continuation of co-pending application Ser. No. 367,008 filedApr. 9, 1982, now abandoned which is a divisional of U.S. Ser. No.041,896, filed May 23, 1979 Pat. No. 4,337,314

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to bacterial vaccines comprisinggenetically-attenuated strains and to processes for their preparation.

2. Description of the Prior Art

The use of bacterial and viral vaccines has been very successful in theprevention of infectious diseases in humans and other animals. There arevarious methods of preparing vaccines from viruses or bacteria. Thebasic requirements for any vaccine and for a method for the preparationof a vaccine are that (1) the resulting vaccine contain the necessaryantigenic determinants to induce formation of antibodies in the host;(2) the vaccine possess high immunogenic potential; (3) the resultingvaccine be safe enough to be administered without any danger of clinicalinfection, either for the recipient or any contact of the recipient,and, therefore, the risk associated with vaccination be minimized, ifnot totally eliminated; (4) the resulting vaccine be devoid of any toxicside-effects, for example, fever from endotoxin present in killed orextracted cells; (5) the resulting vaccine be suitable foradministration by an effective route, for example, oral, intranasal,topical or parenteral; (6) the resulting vaccine and its mode ofadministration mimic closely the circumstances of natural infection; (7)the resulting vaccine be stable under conditions of long-term storage,and that said long-term storage be at room temperature and (8) theresulting vaccine be compatible with the usual inert vaccine carriers.

Prior art methods which have attempted to fulfill one or more of theaforementioned requirements include vaccines containing killed wholecells, purified or partially-purified selected antigenic components, andlive, chemically- or genetically-attenuated microorganisms.

The use of killed whole cells as vaccines has been described by Switzeret al., in U.S. Pat. No. 4,016,253, who applied such a method to thepreparation of a vaccine against Bordetella bronchiseptica infection inswine. Killed whole cells have also been used to prepare a vaccineagainst chronic bronchitis caused by Haemophilus influenzae (Brown andWilson, Br. Med. J. 1: 263, 1959). The use of killed cells, however, isusually accompanied by an attendant loss of immunogenic potential, sincethe process of killing usually destroys or alters many of the surfaceantigenic determinants necessary for induction of specific antibodies inthe host. The antibodies produced in response to such altered antigensare not as specific for the molecular structures on the surface of thelive organism, and, therefore, are not as effective against the invadingpathogen.

Antigenic components, isolated and purified from microorganisms, havealso been used as vaccines. This is represented, for example, by the useof purified capsular polysaccharide material of H. influenzae type b asa vaccine against the meningitis caused by this organism in humans(Parke et al., J. Inf. Dis. 136(Suppl.): S51, 1977; Anderson et al., J.Inf. Dis. 136(Suppl.): S63, 1977; Makela et al., J. Inf. Dis.136(Suppl.): S43, 1977). This approach, however, also suffers fromdrawbacks, in that it requires isolation and purification techniqueswhich may seriously compromise the detailed three dimensional spatialarrangement of the antigen upon freeing it from its position in thewhole cell. The inherent immunogenicity of the antigens extracted fromwhole cells may also be diminished--a loss which may contribute to therelative lack of success with such vaccines in very young children(references supra; Davies, J. Immunol. 33: 1, 1937 and Monto et al., J.Inf. Dis. 127: 394, 1973). Thus, immunization of older children andadults with purified capsular polysaccharide from H. influenzae type bdoes induce humoral antibody, while the very young (less than 14-18months) who are most susceptible to the disease fail to mount asignificant, lasting response (references, supra.) Similar difficultieshave been encountered when purified capsular polysaccharide vaccines areprepared from Streptococcus pneumoniae and Neisseria meningitidis andused to immunize young children (Davies, J. Immunol. 33: 1, 1937; Monto,et al., J. Inf. Dis. 127: 394, 1973).

Chemically-attenuated, live microorganisms have also been used asvaccines in the prior art. This method of preparing vaccines isrepresented, for example, by the work of Wison in U.S. Pat. Nos.3,907,987 and 3,975,517. Wilson prepared a live, bacterial vaccineagainst coliform enteritis in animals from selected strains ofEscherichia coli which were treated with dilute formalin in order torender them less virulent. Bauer et al., U.S. Pat. No. 4,058,599describes the preparation of inactive but immunogenic microorganisms,which were treated with ethyleneimine and therefore rendered lessinfectious. The chemical treatment of whole microorganisms may severelyimpair their immunogenic potential, and in many cases may not decreasevirulence as much as desired.

A second technique for attenuating the virulence of live microorganismswhile allowing them to retain immunogenic potential, is the developmentof avirulent or slow-growing strains, or mutants incapable of sustainedreplication in the host. Such mutants, if well chosen, will maintain thefull integrity of cell-surface constituents necessary for specificantibody induction, yet will be unable to cause disease, because they(1) fail to produce virulence factors, (2) grow too slowly, or (3) grownot at all in the host. A variety of such genetic variants have beenused to prepare bacterial and viral vaccines.

Smith (U.S. Pat. No. 3,364,117), Hillman (U.S. Pat. No. 4,133,875) andGermanier (U.S. Pat. No. 3,856,935) have all described vaccine strainswhich have lost the ability to cause disease presumably because ofmutations in genes responsible for the production of virulence factors.The vaccine described by Smith (supra) comprised a "rough" variant ofSalmonella cholerasuis which was poorly characterized, except for itsloss of virulence in pigs. Since the mutant is rough, the surfaceproperties are altered, and, therefore, not truly representative of theantigens of the pathogenic form. Hillman (supra) described awell-characterized mutant strain of Streptococcus mutans which has asingle point mutation in the structural gene that codes for the enzymeL(+)-lactate dehydrogenase. This mutant strain is a low-acid producerand potentially can replace the normal highacid producing Streptococcusmutans in oral flora in order to reduce the incidence and severity ofdental caries in humans. Germanier (supra) described a mutant strain ofSalmonella typhi carrying a single mutation (characterized as adeletion) which profoundly affects the expression of the genes encodingthe galactose-metabolising enzymes. These genes are clustered in a unit,the gal operon, which encodes the information for the synthesis of threeenzymes--uridine diphosphogalactose-4-epimerase, galactose-1-phosphateuridylyltransferase and galactokinase. The deletion in this strain hasoccurred in the epimerase gene which prevents the production of anyfunctional epimerase enzyme. Characteristically such a deletion alsoexerts strong polar effects on the distal genes of the operon (Martin etal., Cold Spring Harbor Symp. Quant. Biol. 31: 215, 1966) hence thelevels of galactokinase and galactose-1-phosphate uridylyl-transferaseare markedly reduced in this strain. Deletions are usually stable and norevertants of this strain have been detected. Hoever, such galEmutations cause alterations in surface properties by preventing thecomplete formation of the lipopolysaccharide side chains. Hence, thevery structures responsible for inducing type-specific antibodies arecompromised. In addition, galE mutations can result in autolysis,thereby compromising the ability of the vaccine strain to produceprolonged immunological stimulation.

Influenza and respiratory syncytial virus mutant strains which replicateslowly at human body temperature (37° C.) and thus are unable to causedisease, have been prepared (Murphy et al., J. Inf. Dis. 128: 479, 1973and Chanock et al., Pediatr. Res. 11: 264, 1977). These single mutationstrains have proven to produce so many virulent revertants in thevaccinee that they are not practical. There are no reports ofslow-growing bacterial mutant strains appropriate for use as vaccines,but the same limitations which apply to the slow-growing viral strainswould also apply to them.

Two classes of mutants have been used to prepare vaccine strains whichdo not replicate at all in the host. The first class includes strainswhich are dependent upon unusual nutrients or growth factors (compoundsoutside the biochemistry of the vaccinee) for replication as describedby Levine (J. Inf. Dis. 133: 424, 1976) and Reitman (J. Inf. Dis. 117:101, 1967). These workers described strains of Salmonella typhi andSalmonella typhosa which require streptomycin for growth. Presumably thestructure of ribosomal proteins in these strains is modified so thatstreptomycin is required for proper ribosome function. These strains,however, do not have sufficient genetic stability to make them trulysafe vaccines.

The second class includes strains which are incapable of replicationabove certain temperatures as described by Fahey and Cooper (Infect.Immun. 1: 263, 1970), Maheswaran (U.S. Pat. No. 3,855,408) and Helms etal., (J. Inf. Dis. 135: 582, 1977). Temperature-sensitive strains ofSalmonella enteritidis, Pasteurella multocida and Streptococcuspneumoniae were isolated and partially-characterized by these workers.The strains were unable to grow at the body-temperature of the animalsthey were tested in, but were excellent immunogens, since the surfaceproperties of the cells were unaltered. Significant reversion tovirulence, however, has precluded their use in humans and other animals.

The reversion problem associated with attempts to usetemperature-sensitive strains as vaccines appears to be an inherentlimitation of the approach. This is because ts mutations result fromsingle base changes and therefore exhibit significant spontaneousreversion. Similarly, reversion (to virulence) has also posed a problemin the safety of employing genetically-attenuated viral strains asvaccines (Henderson et al., J.A.M.A. 190: 41, 1964; Chanock et al.,Pediatr. Res. 11: 264, 1977 and Murphy et al., J. Inf. Dis. 128: 479,1973)

In order to overcome this problem, the prior art has investigated thevaccine potential of virus strains which contain two growth-attenuatingmutations (Murphy et al., Virology 88: 231, 1978 and Murphy et al.,Infect. Immun. 23: 249, 1979). In such strains, two mutations ofdifferent phenotype were combined with the expectation that thereversion frequencies would decrease.

Two methods have been used to combine multiple ts mutations in onestrain. First, Chanock et al., (Pediatr. Res. 11: 264, 1977) mutagenizeda strain of respiratory syncytial virus which already contained a tsmutation, and isolated a strain which contained a second ts lesion. Thefirst mutation almost completely blocked virus replication at 39° C.;the second mutation severely limited replication at 37° C., and so thedouble mutant strain could be recognized.

A second method using natural in vivo recombination was reported byMurphy et al., (Virology 88: 231, 1978). They constructed a doubly tsstrain of influenza virus by co-infecting cells withtwo virus strains,one containing a mutation with a cut-off temperature of 37° C. and thesecond containing a mutation in another gene with a cut-off temperatureof 38° C., and isolating recombinant viruses among the progeny. Therationale for preparation of these strains was that the resultingisolates would contain two growth restricting mutations, the originalmutation which severely restricts replication at 38° or 39° C. and asecond mutation which severely restricts replication at or above 37° C.At temperatures of 39° C. or above both mutations severely limit virusreplication; and revertants are extremely rare (as outlined above).However, at 37° C., normal body temperature, only one of the twomutations will limit growth and the virus will phenotypically expressonly one mutation; resulting in a reversion frequency equal to that ofthe 37° C. mutation alone. Double-ts mutant strains, where thetemperature cut-off points are not the same, can also producesignificant numbers of revertants of one of the ts mutations if they arepropagated at temperatures near the lower cut-off temperature. Suchtemperatures are semi-permissive and provide strong selective pressurefor overgrowth of the original strain by revertants.

While the use of multiple mutations has not been applied to bacterialvaccine strain preparation, Curtiss has constructed a strain ofEscherichia coli which is attenuated by the incorporation of multiplemutations of different phenotype, for gene cloning (Ann. Rev. Microbiol.30: 507, 1976). The important consideration in this work was to producea strain that could not survive outside a specialized environmentprovided in the laboratory. In fact, at least one of the mutationsintroduced into the strain causes the self-destruction of the cell whenit attempts to grow outside the defined specialized environment. Curtisshas suggested that this strain could be used to clone the gene(s) for abacterial toxin which could then be produced in bulk for vaccine use(ibid, at 510, lines 26-28). Furthermore, British Patent SpecificationNo. 1-516-458, published July 5, 1978 in the United Kingdom, describesthis microorganism, Escherichia coli K-12 _(x) 1776 and the manymutations which have been introduced into it in order to make it safefor the large-scale production of prokaryotic or eukaryotic proteins(for example, cholera toxin or insulin), or organic acids (for example,succinic or lactic acid produced by fermentation of glucose). Thespecification describes on p. 4 the use of microorganisms which havebeen precluded from growth or colonization in natural ecological niches.However, the main thrust of the specification is directed towards theconstruction of bacterial strains containing various mutations conveyingmany different phenotypes in one strain. The disadvantage of using suchan approach for construction of vaccine strains are discussed below.

The combination of two mutations of different phenotype in a singlestrain will only be effective in reducing the revertant frequency whenboth mutations are simultaneously restrictive. This argument is equallyapplicable to temperature-sensitive, growth factor or virulence factormutations, alone or in combination. Ideally, to achieve reducedreversion frequencies by combining two or more mutations in a singlestrain, the mutations must convey identical phenotypes so as to ensurethat they always work in concert.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a safe vaccinebased on live, genetically-attenuated bacterial microorganisms. It isanother object of the invention to provide vaccines based on bacterialstrains carrying two or more mutations.

It is a further object of the invention to provide bacterial vaccinesbased on strains which contain two or more mutations of the samephenotype.

Still another object of the invention is to provide bacterial vaccinesbased on strains which contain two or more temperature-sensitivemutations which have an identical cut-off or restrictive temperature.

Still a further object of the invention is to provide bacterial vaccinesbased on strains which maintain antigenic integrity and remain intactafter administration.

An object of the invention is to provide bacterial vaccines based onstrains which are capable of at least limited replication in the host,in order to simulate the initiation of normal infection.

Still a further object of the invention is to provide a bacterial strainwhich is suitable as a vaccine for Haemophilus influenzae per se, and,by genetic addition of appropriate capsular information, vaccines forany and all serotypes of Haemophilus influenzae.

An object of the invention is also to provide a method of inoculationagainst infectious diseases by using genetically-attenuated bacterialvaccine strains which contain two or more mutations of the samephenotype.

Another object of the invention is also to provide a process for thepreparation of genetically-attenuated vaccine strains containing two ormore mutations of the same phenotype.

These and other objects of the invention as hereinafter will become morereadily apparent have been attained by providing a vaccine comprising agenetically-attenuated bacterial strain which contains two or moremutations of the same phenotype which strain is rendered avirulent whileretaining immunogenicity.

The objects of the invention have also been attained by providing avaccine wherein the two or more mutations of the same phenotype aretemperature-sensitivity mutations of the same phenotype.

The objects of the invention have also been attained by providing avaccine against meningitis and other diseases caused by the organism,which comprise a genetically-attenuated strain of Haemophilus influenzaewhich contains at least two different ts mutations of the samephenotype.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein

FIG. 1 describes the protocol used for isolating ts mutants of H.influenzae.

FIG. 2 describes the general strategy employed for locating andcharacterizing ts lesions in mutated strains of H. influenzae.

FIG. 3 describes the growth curves of the multiple-mutant strains,A214-A/3-C2/13 (O) and the wild-type, 001 (□), at 29° C.

FIG. 4 describes the growth curves of the multiple-mutant strain,A214-A/3-C2/13 (O) and the wild-type, 001 (□), after temperature shiftfrom 29° C. to 34° C.

FIG. 5 describes the growth curves of the multiple-mutant strain,A214-A/3-C2/13 (O) and the wild-type, 001 (□), after temperature shiftfrom 29° C. to 36° C.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The essential properties of vaccines are that they: (1) effectivelyinduce protective immunity in the vaccinee, (2) do not produce diseaseand (3) do not have toxic side-effects. Other desirable properties ofvaccines include: (1) inability to establish carrier-states forcommunicable pathogens, (2) inexpensive and simple preparation, (3)stability and (4) ease of administration. The vaccine preparationsdescribed in this invention meet all these requirements, for they: (1)use whole cells whose surface antigens are identical with those of thepathogen against which immunity is sought; hence they possess highimmunogenic potential, making low doses effective and minimizingdose-dependent toxic side-effects, (2) use genetically-attenuated, livecells which cannot cause disease in the vaccinee, (3) are essentiallyfree of virulent forms of the pathogen (less than one virulent cell persextillion [10²¹ ] vaccine organisms) and (4) can be cultured, storedand administered by standard microbiological and immunologicalprocedures.

Specifically, this invention describes a method for the preparation ofsafe, effective, live vaccine strains. This has been accomplished bycombining multiple attenuating mutations of identical phenotype intootherwise normal pathogenic strains, thus rendering the strain avirulentin the normal host.

ADVANTAGES OF LIVE, GENETICALLY-ATTENUATED VACCINES

One advantage of working with live, mutant microorganisms, as opposed toisolated antigenic components, is that it ensures, at least formutations which do not affect cell-surface and extracellular antigeniccomponents, the presence of all essential structural features necessaryfor succesful induction of antibody synthesis in the host. It is wellestablished that the detailed three-dimensional arrangement of theantigen-combining site is highly specific and necessary for molecularrecognition of antigen by antibody. (Kabat, Structural Concepts inImmunology and Immunochemistry, Holt, Rinehart and Winston, Inc., 1968;chapter 6). If the three dimensional molecular arrangement of theantibody-inducing vaccine is not as close as possible to the moleculararrangement of the antigens of the infecting microorganism, then theantibodies formed against the inducing vaccine will not be verysuccessful against the challenger. In other words, the avidity of theseantibodies for the "natural" antigens will be low. If the vaccine isantigenically identical with the microorganism which will pose theeventual challenge, the avidity of the antibodies formed against thevaccine, and therefore, also against the antigens of the pathogen iscorrespondingly very high.

The vaccines of the present invention also embody the specific advantageof maintaining the inherent adjuventicity of whole cells. Thepotentiation of the immunogenicity of certain antigens (for example,polysaccharides) by other cellular components is well-documented. Thepresent invention exploits this property maximally.

A further specific advantage of the present invention is that vaccinesprepared from strains with the appropriate attenuating mutations, cangrow in the recipient and mimic natural infection.

SPECIFIC FEATURES OF THE INVENTION

The most important element of this invention is that the microorganismshave multiple mutations of the same phenotype in the same strain. Thisfeature creates a protective guard against reversion of the mutatedstrain to the virulent form. It also provides a solution to a longrecognized need in the art of genetically-attenuating bacterialvaccines. The genetic instability of viral and bacterial mutants haslong been one of the most serious obstacles to the widespread use ofgenetically-attenuated strains as vaccines. The present invention hasaccomplished a breakthrough in this respect and opens the door to theextensive use of mutant strains as vaccines. It overcomes the problem ofreversion in mutated strains by reducing the effective reversionfrequency of such strains to negligible levels (<10⁻²⁰). This has beenaccomplished by incorporating multiple, independent mutations whichconvey the same phenotype, into a single strain. The two or moreindependent mutations used render the strain incapable of causingdisease in the host. The reversion rate of a strain containing multiplemutations of the same phenotype is the product of the reversionfrequencies of the individual mutations. If three mutations, each with areversion frequency of ca. 10⁻⁷, are combined, a strain with a reversionrate of 10⁻²¹ is obtained. This means that if every man, woman and childpresently on earth and all future generations were immunized with such avaccine, using a dose of one million organisms per vaccinee, only oneindividual in the next ten million years (10¹⁵ people) would be exposedto a virulent revertant!.

CHOICE OF ATTENUATING MUTATIONS

An important aspect of the solution to the reversion problem presentedin this invention, is that all the mutations affect functions whichcannot be corrected under the normal physiological conditions of thehost. Potentially, such mutants include (1) those that do not producevirulence factors in the host, (2) those dependent upon the presence ofgrowth factors not normally found in the host and (3) those unable tosustain replication at the body temperature of the host.

The first group, those which do not produce virulence factors in thehost, can be defined as mutants unable to make functional virulencefactor(s) under any conditions or under the physiological conditions ofthe host. Such mutants, for example, may only produce non-toxic, butimmunogenic fragments of a toxin, may produce no toxin at the bodytemperature of the host, or may be incapable of producing toxin underany condition. Such mutants would also include strains unable to producefactors necessary for tissue invasion. Such mutants would also includestrains unable to make pili required for adherence to animal cells(colonization factors), although it should be recognized that suchvaccine strains may thereby suffer loss of important antigens and theability to persist in the area necessary to alert the immune system.

The second group can be divided into two classes. First, those mutantsunable to synthesize intermediate metabolites unique to procaryoticorganisms. One example is diaminopimelic acid (DAP), a compound notfound in the biochemistry of eukaryotic organisms, but which is anessential intermediate in the biosynthesis of cell-wall material in manybacterial species. In the absence of DAP such strains lyse as a resultof cell growth. Second, those mutants that require certain compoundswhich are not metabolized but are essential for growth. Mutant strainswhich require streptomycin fall into this class.

The third group, temperature-sensitive (ts) mutants, contain lesionswhich do not permit sustained replication of the organism at the bodytemperature of the host. When these lesions directly affect essentialfunctions, such as transcription, translation or transport, they cannotbe corrected by any environmental factor other than temperature. Hence,temperature-sensitive mutations which affect essential functions have adistinct advantage over growth-factor dependent mutations for there isalways the chance that unique microbial metabolites could be supplied byhost flora or iatrogenic intervention.

Specifically, temperature-sensitive mutations can be divided into fourclasses: (1) those that cause growth to terminate "immediately" ontransfer to the non-permissive temperature; (2) those that restrict thegrowth rate at the non-permissive temperature; (3) those that grow andlyse, and (4) those that have little or no immediate effect on growth,but terminate growth following a number of cell-divisions at therestrictive temperature. The first class allows precise control over thenumber of organisms used to inoculate the individual vaccinee, but sincegrowth is "immediately" terminated following inoculation, theimmunogenic potential of the vaccine may be compromised because earlysteps in the disease process cannot be mimicked. The second class of tsmutations, those which allow slow growth at the restrictive temperature,can provide long-term exposure to the microbial antigens but, since suchstrains can replicate ad infinitum at the restrictive temperature, thereis no way to eliminate the possibility that the vaccinee will be exposedto virulent revertants. The third class of ts mutations, those whichbring about lysis at the restrictive temperature, may cause removal ofthe vaccine organisms before the immune system has a chance to respondto their presence. The fourth class of ts mutations, those whichinitially allow "normal" replication after transfer to the restrictivetemperature, but completely block sustained replication in the vaccinee,can be used to produce vaccine strains which can, upon inoculation,initiate normal infective processes, but which cannot cause disease.

The most essential feature of the approach herein presented is that allthe mutations in a given strain convey the same phenotype. This featureis important in that it ensures that all of the mutations incorporatedwork in concert. Hence, the effective reversion frequency is guaranteedalways to be the product of the reversion frequencies of the individuallesions. To further elaborate this point the bacterial vaccines of thepresent invention carry multiple (i.e., two or more) mutations, witheach independent mutation conveying the same phenotype. Thus, in oneembodiment of this invention a bacterium may, for example, carry two 37°C. ts lesions, ts₁ and ts₂. The resulting microorganism is unable togrow at 37° C. or higher temperatures. Without being bound by anyparticular theory, Applicants suggest that such a bacterium probably hastwo different mutations which affect two different proteins essentialfor growth, protein₁ and protein₂. In the multiple mutant strain neitherprotein is functional at 37° C. or higher temperatures, but bothproteins are functional at lower temperatures. If the ts₁ mutationshould spontaneously revert, the protein₁ produced will be functional at37° C. Since ts₂ has not reverted, however, protein₂ remainsnon-functional at 37° C. and hence the growth and replication of therevertant is still restricted at 37° C. Not until ts₁ and ts₂ have bothreverted will the cell produce functional protein₁ and protein₂, andregain the capacity to grow at 37° C. The same model may be applied toother mutations, such as antibiotic-dependent mutations or the like. Thefulfilment of this requirement (identical phenotypes) may be practicallydifficult, for example, with multiple streptomycin-dependent mutationswhen all the lesions would have to reside in the same gene. Not onlywould such a strain be technically difficult to construct, but also, inthe event of in vivo genetic exchange, incorporation of one small pieceof DNA could eliminate all the lesions. There is no such restriction ontemperature-sensitive mutations, for multiple lesions can be widelydistributed in the genetic material. Hence, the probability forcorrection of the lesions is reduced by the product of the probabilitiesfor in vivo genetic correction of each individual lesion. Theprobability of this happening (in vivo genetic recombination) withwidely separated mutations is so low as to not to constitute a problem(Curtiss, Ann. Rev. Microbial. 30: 507, 1976).

ISOLATION OF MUTANT STRAINS

Strains containing attenuating mutations can originate either asspontaneous derivatives or after mutagenic treatment. Not only canmutagenic treatment be applied to whole organisms but also to DNA whichcontains relevant genetic information (provided at the DNA issubsequently incorporated into the whole organism). When the mutationconfers a selectable phenotype, the mutant derivatives can be isolatedand identified by standard genetic techniques. Mutations which confer aphenotype only detectable by specialized techniques can be identified byemploying those techniques (for example, see Maas et al., Proc. Nat.Acad. Sci. 75: 1384, 1978). Detailed descriptions for the isolation ofmutant strains whose replication is temperature-sensitive are presentedin the next section.

CONSTRUCTION OF MULTIPLE MUTANT STRAINS

An important feature of this invention is that the multiple mutationswhich are combined into a vaccine strain convey the same phenotype. Thefact that each mutation exhibits an identical phenotype precludes directselection of multiple mutant strains, for there is no simple way todistinguish between single and multiple mutant isolates. We have solvedthis problem by isolating individual mutant strains containing singleattenuating mutations, and then transferring these mutations into asingle strain (by genetic transfer and recombination). To facilitate thedetection of strains which have incorporated the desired attenuatingmutation(s) each attenuating mutation used is genetically linked to areadily selectable and scorable "non-attenuating" allele. The linkedmarkers are selected in the strain constructions and then thesimultaneous incorporation of the desired attenuating mutation(s) testedamong the recombinants.

Two classes of phenotype lend themselves to exploitation for such straincontruction: (1) auxotrophy--a strict requirement for an amino acid ornutritional factor, and (2) chromosomal resistance--mutations whichconvey resistance to selected agents (e.g. antibiotics, colicins, orbacteriophage).

Mutants representing both the above phenotypes can be isolated in astraightforward manner and used for positive genetic selection (e.g.prototrophy--the requirement for a nutritional factor is "cured", orresistance--the ability to grow in the presence of a bactericidalagent). Attenuated mutants are isolated from auxotrophic or wild-typestrains. Appropriate selectable markers are introduced as part of themapping procedure. Strains with attenuating mutations linked toselectable markers are used for the subsequent constructions.Recombinants which have potentially incorporated the attenuatingmutations are first identified by selection of the linked selectablemarker. Successful concomitant transfer of the attenuating mutation isconfirmed by recovery of the mutation from the recombinant (see thefollowing section for a detailed description of such procedures).

MECHANISMS FOR GENETIC TRANSFER AND RECOMBINATION

There are four recognized mechanisms for transfer of genes (DNA) fromone bacterial cell to another which may be used to prepare the vaccinesof this invention: (1) transformation naked DNA from cell "A" is takenup by cell "B" and incorporated into the chromosome of cell "B"(Alexander and Leidy, J. Ex. Med. 97: 17 1953); (2) transduction--virusparticles containing DNA from cell "A" attach to cell "B" and "inject"the DNA into cell "B", where it is incorporated into the chromosome ofcell "B" (Lennox, Virology 1: 190, 1955; (3) conjugation--cell "A" andcell "B" come together and DNA from cell "A" passes unidirectionallyfrom cell "A" into cell "B" and is incorporated into the chromosome ofcell "B" (Wollman et al., Cold Spring Harbor Symp. Quant. Biol. 21: 113,1964) and (4) fusion--cells "A" and "B" are chemically treated, fusionof the two cells occurs and DNA from "A" and "B" transiently reside inthe same cell before recombination occurs (Fodor and Alfodi, Proc. Natl.Acad. Sci. 73: 2147, 1976). In vitro recombination of geneticinformation can be accomplished by isolation of DNAs, enzymatic cleavageand insertion of "A" into "B", and then transformation of cells with thehybrid DNA. Any or all of these mechanisms may be used to move genesamong bacteria.

Other methods to prepare the bacterial vaccines of the present inventionare in vitro mutagenesis of specific DNA molecules (Bautz-Freese andFreese, Virology 13: 19, 1961) de nova gene synthesis (Khorana, Science203: 614, 1979) and construction of mutated genes by recombinant DNAtechnology. Although such methods are in their infancy, it is possibleto foresee the selective and specific preparation of a gene with two ormore well-defined base changes at the DNA level. Such awell-characterized gene can then be reintroduced into the bacterialchromosome and thus result in the creation of a well-defined mutant. Ifthe two or more base changes are chosen so as to produce two or moredifferent mutations of the same phenotype, then the resulting bacteriumwill be encompassed by those of the present invention.

ADVANTAGES OF THE MASTER STRAIN

One of the major advantages of the present invention is that thetechniques can be applied to introduce multiple mutations of the samephenotype into a "rough" strain of, for example, Streptococcuspneumoniae, and then the capsular genes for each clinically-significantserotype (and there are at least 14) can be transformed into the"master" strain to produce a vaccine strain for each. In fact, themultiple-mutant strain of the present invention is a "rough" variant ofH. influenzae, and it is anticipated that this strain can not only beused as a vaccine to protect from infection with such "untypable" H.influenzae (for example, most cases of otitis media in young childrenare caused by "rough" H. influenzae) but it can also be modified bytransformation with the capsular genes for the b serotype, making itsuitable as a vaccine to protect from influenzal meningitis.

STORAGE

One of the advantages of the present invention is that the vaccine canbe easily prepared, lyophilized in the presence of appropriate inert,non-toxic carrier(s) (infra) in vials and stored at room temperaturewithout loss of potency. No refrigeration or special storage equipmentis required.

QUALITY CONTROL

The composition of vaccine preparations must be known and consistent.This is achieved by using specified amounts of quality-controlledchemical and biological ingredients in their preparation. Methods forthe quality control of chemical components are well established in theart and will not be discussed here. Chemical purity in the vaccinepreparations is defined as freedom from toxic waste or cellularbreakdown products and interfering or spurious immunogenic material.This is assured by working with pure cultures (the vaccine strain freeof other cells or virus) and harvesting the cells while the culture isin the logarithmic phase of growth (before the synthesis of autolyticenzymes). The collection and washing of cells from the medium byphysical methods (centrifugation) should leave low molecular weightimpurities in the supernatent.

The present invention has certain advantages with respect to qualitycontrol of the biological components of the vaccine. Standard techniquescan be used to monitor the general biological purity of preparations(freedom from contaminating virus or bacteria). The specific advantagesof this invention relates to the second level of biological purity whichmust be maintained--genetic purity. The most important factor formaintenance of genetic purity in the propagation ofgenetically-attenuated vaccine strains is the choice of attenuatingmutations. It is essential that such mutations do not limit the rate orextent of growth of the strains under all conditions used formaintenance, pilot, or large-scale production. This ensures that whenrevertants do arise they will not possess any selective growth advantageover the vaccine strain, and hence will not outgrow (and in so doingreplace) the vaccine strain in any phase of their culture underpermissive conditions. The appearance of revertants can be monitored byplating a portion of culture under non-permissive conditions. Themaintenance of the attenuating mutations incorporated in the vaccinestrain can be checked by rescue of those mutations from a sample ofcells taken from a vaccine preparation.

ADMINISTRATION

The vaccines of the present invention can be administered to any warm-or cold-blooded animals susceptible to infection with pathogenicmicroorganisms. Human and non-human animals may benefit as hosts.

Administration can be parenteral, but preferably oral or intranasal,depending upon the natural route of infection. In farm animals, forexample, the vaccine may be administered orally by incorporation of thevaccine in feed or feed water. The dosage administered may be dependentupon the age, health and weight of the recipient, kind of concurrenttreatment if any, and nature of the organism. Generally, a dosage ofactive ingredient will be from about 10¹ to 10¹⁰ cells per applicationper host. The preferred dose for intranasal administration wouldgenerally be about 10⁶ organisms, suspended in 0.05 to 0.1 ml of animmunologically inert carrier. Peroral administration of a vaccinestrain of, for example, Salmonella typhi developed according to themethod described in this invention would probably require 10⁶ to 10⁸organism suspended in 1-2 mls of, for example, skim milk. The vaccinescan be employed in dosage forms such as capsules, liquid solutions,suspensions, or elixirs, for oral administration, or sterile liquid forformulations such as solutions or suspensions for parenteral, intranasalor topical (e.g. wounds or burns) use. An inert, immunologicallyacceptable carrier is preferably used, such as saline, phosphatebuffered saline or skim milk.

APPLICATION

Any microorganism capable of producing infectious disease can begenetically-attenuated according to the methods of the present inventionto yield a useful and safe vaccine. Among these, bacteria, viruses andparasites are the most common microorganisms. The present invention isparticularly concerned with bacterial mutations because of the ease ofconstructing the desired (recombinant) mutant strains. Table 1 listscommon bacterial infections and the microorganisms which cause them. Thelist is not designed to be all inclusive but simply exemplary of thelarge number of bacteria which may be utilized. The hosts may be anyhuman or non-human animal.

Having now generally described this invention, a more completeunderstanding can be obtained by reference to certain specific examples,which are included for purposes of illustration only and are notintended to be limiting unless otherwise specified.

                  TABLE 1                                                         ______________________________________                                        Some Common Bacterial Infections                                              Disease           Bacterium                                                   ______________________________________                                        Meningitis        Haemophilus influenzae                                                        Neisseria meningitidis                                      Tuberculosis      Mycobacterium tuberculosis                                  Fowl cholera      Pasteurella multocida                                       Pertussis         Bordetella pertussis                                        Plague            Pasteurella pestis                                          Anthrax           Bacillus anthracis                                          Septicemia, pneumonia,                                                                          Pseudomonas aeruginosa                                      Typhoid fever     Salmonella typhi                                            Pneumonia         Streptococcus pneumoniae,                                                     Mycoplasma pneumoniae,                                                        Staphylococcus aureus                                       Coliform enteritis                                                                              Escherichia coli                                            Dental caries     Streptococcus mutans                                        Cholera           Vibrio cholerae                                             Gonorrhea         Neisseria gonorrheae                                        ______________________________________                                    

PREPARATION OF A VACCINE AGAINST H. INFLUENZAE INFECTION Background

Haemophilis influenzae type b is the major cause of endemic meningitisin children (Haggerty and Ziai, Adv. Ped. 13:129, 1964; Wehrle et al.,Pediatrics 44:991, 1968; and Smith and Haynes, Pediatrics 50:723, 1972)and a significant cause of fatal epiglottitis, obstructivebronchiolitis, otitis media, septic arthritis, laryngitis, cellulitisand pneumnomia in both chlidern and adults. H. influenzae also causesmeningitis in adults. At least 10,000 cases of meningitis due to H.influenzae type b occur annually in the United States. Rapid diagnosisand treatment with ampicillin or chloramphenicol usually leads torecovery. The mortality rate, however, has remained at 5-10%, and asignificant number (30-50%) of those who do recover, suffer permanentneurological damage.

The recent emergence of a plasmid mediating drug resistance in H.influenzae type b has compromised antibiotic therapy in those areaswhere it has appeared (Center for Disease Control, Morbidity andMortality Weekly Rep., 23:77, 1974). The precedent of the rapiddissemination of such plasmids worldwide indicates that within a shorttime antibiotics will no longer be effective in the control of H.influenzae infections. The specter of totally antibiotic-resistant H.influenzae type b together with the high incidence of serious permanentneurological sequelae make it imperative to develop new methods tocontrol infections by this organism. Currently available purifiedpolysaccharide vaccines for H. influenzae are not effective in veryyoung children--those most likely to succumb to the organism. The needfor an effective, safe vaccine against H. influenzae infection istherefore acute.

A recombinant strain of H. influenzae containing three ts mutationsconveying the same phenotype was prepared using transformtion asdescribed in the present invention.

Materials and Methods

The following abbreviations are used throughout the followingdescription of the preparation of a vaccine against H. influenzaeinfection.

A/b, antibiotic

BHI, brain heart infusion

cfu, colony forming unit

DMSO, dimethyl sulfoxide

DNA, deoxyribonucleic acid

Em^(R),S, erythromycin-resistant, --sensitive

g, gram Km^(R),S, kanamycin-resistant, --sensitive

l, liter

mg, milligram ml, milliliter

NAD, nicotinamide adenine dinucleotide

Nal^(R),S, naladixic acid-resistant, --sensitive

Nb^(R),S, novobiocin-resistant, --sensitive

NG, nitrosoguanidine, N-methyl-N'-nitro-N-nitrosoguanidine

PRP, polyribophosphate

Rif^(R),S, rifamycin-resistant, --sensitive

RR, reversion rate

Sm^(R),S, streptomycin-resistant, --sensitive

ts, temperature-sensitive

u, micron

ug, microgram

ul, microliter

Vm^(R),S, viomycin-resistant, --sensitive

Bacteria. Two basic strains of H. influenzae were used in this work: therough type d strain (strain 001) and a derivative resistant to theantibiotics, Em, Km, Nb, Sm, Vm and Nal (strain EKNSVNal). Strain 001(ATCC#31517) and EKNSVNal both exhibit the characteristics described forH. influenzae in Bergey's Manual of Determinative Bacteriology, (7thedition, 1957) pp. 406-408--they are gram-negative rods, 0.2-0.3 by0.5-2.0 microns, occurring singly and in pairs, occasionally in shortchains. They require the growth factors nicotinamide adeninedinucleotide and hemin. EKNSVNal has been deposited at the ATCC and hasthe #31514.

Growth Media. H. influenzae was passaged routinely on brain heartinfusion (BHI) (2.5% w/v) agar supplemented with hemin (20 ug/ml) andNAD (10 ug/ml) or chocolate agar. Liquid cultures were grown insupplemented BHI broth. Selective platings were on supplemented BHI agarcontaining the appropriate antibiotic.

Mutagenesis, Enrichment and Isolation. The protocol followed forisolating ts mutants of H. influenzae (001 and EKNSVNal) is outlined inFIG. 1. Since it may be necessary to colonize the upper respiratorytract with the vaccine strain, at least transiently, in order to alertthe immune system, two types of ts mutant were sought. The classical"tight" mutant which ceases replication immediately after transfer tothe non-permissive temperature was routinely isolated by following theprocedure described in FIG. 1. Mutants which "coast" for two or threegenerations after transfer to the restrictive temperature were isolatedby delaying addition of antibiotic. Mutants with cut-off temperatures of32°-36° C. were also sought by varying the temperatures at which theenrichments were performed.

Preliminary Characterization of Mutants. The ts mutants isolated by theprocedure described above were tested to determine temperature cut-offpoints by streaking on chocolate agar plates and incubation at 27°, 30°,32°, 34° and 36° C. Those mutants which grew along the primary streakbut which were unable to form single colonies at the restrictivetemperature were designated "coasters". Classical "tight" mutants wereof course unable to grow at all above their temperature cut-off point.

The reversion rates of the individual mutant strains were determined byspreading 1-2×10⁹ cells on chocolate agar plates which were incubated atthe restrictive temperature. "Coasters" in liquid culture were alwaysincubated at the restrictive temperature for 1 hour before plating.

The ability of "coasters" to continue growth after transfer to thenon-permissive temperature was tested in liquid culture and "coasting"and final arrest monitored by absorbance at 600 nm and quantitation ofcolony-forming units.

Genetic Mapping. Location of the ts lesions on the H. influenzaechromosome was accomplished by a series of transformations. The generalstrategy employed is outlined in FIG. 2. DNA from strain EKNSVNal wasused to transform the ts 001 derivatives and transformants were selectedon supplemental BHI agar containing the appropriate antibiotic.Antibiotic-resistant recombinants were then streaked on duplicate plateswhich were incubated at the permissive and non-permissive temperatures.If the ts lesion in an 001 mutant was "cured" by the acquisition of agene conferring, for example Sm resistance, then that lesion waspresumed to be linked to the Sm resistance gene.

The inverse experiment was also always performed--EKNSVNal DNA was usedto transform ts 001 strains and the ts⁺ phenotype selected. The ts⁺recombinants were then scored for the acquisition ofantibiotic-resistance genes by replica-plating on supplemented BHI agarcontaining the appropriate antibiotics.

Similarly, ts EKNSVNal mutants were transformed with 001 DNA andrestoration of the ts⁺ phenotype in the recombinants for the concomitantloss of antibiotic-resistance. DNA from ts EKNSVNal strains was used totransform 001, antibiotic-resistance selected for at 27° C. andrecombinants screened for temperature-sensitivity.

Transformation. The plate technique developed by Juni (Appl. Microbiol.27:16, 1974) and modified by Clark et al., (Abstracts, A.S.M. AnnualMeeting, 1977) was used for all transformations after preliminaryexperiments determined that the method was efficient at temperatures aslow as 27° C. DNA was prepared by lysing cells (1-2×10⁹ /ml) suspendedin supplemented BHI broth containing 0.02% SDS and the preparationsterilized by heating at 60° C. for 15 minutes. Overnight plate culturesof the recipient bacteria were smeared on fresh chocolate agar andthoroughly mixed with one drop of DNA delivered from a 0.1 ml pipette.The plates were incubated for 3 hours (when ts⁺ recombinants weresought) or 7-18 hours (when antibiotic-resistant transformants weredesired) at 30° C. and transformants streaked on chocolate agar orsupplemented BHI agar containing the appropriate antibiotic. Thoseplates were then incubated at 36° C. in the former case and at 27° C. inthe latter.

Purification of Mutant Alleles. Although NG is an excellent mutagen itstendency to induce additional mutations, both closely linked and distantfrom the gene of interest (Adelberg et al., Biochem. Biophys. Res. Comm.18:788, 1965 and Hirota et al., J. Mol. Biol. 35:175, 1968), can causeproblems. It is therefore desirable that, once the selected mutation hasbeen deemed suitable it be transferred from the mutagenized "dirty"strain to a "clean" background. Accordingly, when the ts lesions hadbeen mapped and linkage established to antibiotic-resistance markers,the genes were transformed into 001. DNA from the ts mutants generatedin EKNSVNal was used to transform 001 and the transformants plated onsupplemented BHI agar containing the appropriate antibiotic.Antibiotic-resistant recombinants were then screened for the presence ofthe ts gene. The reversion rates of the ts mutation were confirmed.Those ts derivatives of 001 whose lesions had been located near anantibiotic-resistance gene were first rendered antibiotic-resistant bytransforming the appropriate marker from the "clean" EKNSVNal into thestrain and monitoring for retention of temperature-sensitivity. TheA/b^(R) -ts linked gene was then transformed into the "clean" 001strain. In this way both sides of the antibiotic-resistance linked tsgenes were purged of "contaminating" mutations induced bynitrosoguanidine treatment.

Final Strain Construction. The 001 strains carrying the "clean" ts geneswere combined by transformation, using the appropriate antibiotics forselection. Double recombinants were tested first for a reduction inreversion rate. The presence of the second ts gene was confirmed byrecovering it in a second transformation to a ts⁺ strain. Triplerecombinants could only be tested by recovery of the three ts genes inseparate transformations.

Results

Single Mutant Isolates. Following the scheme outlined in FIG. 1 threestrains which contained "coaster" ts mutations linked to selectablemarkers and which exhibited appropriate reversion frequencies andcut-off temperatures were selected. Their properties are listed in Table2.

                  TABLE 2                                                         ______________________________________                                        MUTANT DERIVATIVES OF STRAINS                                                 001 AND EKNSVNal                                                                        Temp.                Reversion                                      Number    cut-off     Linkage  Rate                                           ______________________________________                                        A/3       34° C.                                                                             Km.sup.R 6 × 10.sup.-8                            A214      34° C.                                                                             Sm.sup.R 1 × 10.sup.-8                            C2/13     34° C.                                                                             Em.sup.R 1 × 10.sup.-7                            ______________________________________                                    

Recombinant Strain. The three ts lesions were combined in a singlestrain by sequential transformations. A214 was first transformed withA/3 DNA, Km^(R) recombinants were selected and analyzed for loweredreversion frequencies. The presence of the ts lesion linked to Km^(R)was confirmed by using DNA from the recombinant to transform 001.Maintenance of the ts lesion linked to Sm^(R) was confirmed in a similarway. DNA from strain C2/13 was used to transform the recombinantA214-A/3 strain to Em^(R). The presence of all three ts lesions wasconfirmed by recovering them in separate transformations of 001 (asdescribed above). The reversion rates of these strains are listed inTable 3.

Both the double and triple recombinant strains have been deposited withthe American Type Culture Collection, strain #'s 31515 and 31516respectively. The taxonomy of the strains is identical with the startingstrains, with the following modifications: growth is optimal at 29° C.,inhibited at 34° C. and limited to two divisions at 36° C.; the strainis resistant to the antibiotics kanamycin, streptomycin anderythromycin.

                  TABLE 3                                                         ______________________________________                                        PARTENTAL AND RECOMBINANT STRAINS                                                           Reversion Rate                                                  Strain          Observed   Calculated                                         ______________________________________                                        A214            1 × 10.sup.-8                                                                      --                                                 A/3             6 × 10.sup.-8                                                                      --                                                 C2/13           1 × 10.sup.-7                                                                      --                                                 A214-A/3        <3 × 10.sup.-9                                                                     6 × 10.sup.-16                               A214-A/3-C2/13  <3 × 10.sup.-9                                                                     6 × 10.sup.-23                               ______________________________________                                    

Encapsulation of the Master Strain. The recombinant strain constitutes amaster strain which is suitable for use as a vaccine against untypablestrains of H. influenzae and, by transformation with DNA encoding thegenes responsible for capsule formation, can be modified to producevaccine strains against any and all of the six serotypes of H.influenzae. This modification of the master strain can be accomplishedby preparing DNA from an encapsulated strain, using the DNA in atransformation mixture as described above, and plating the recombinantson supplemented BHI agar containing antiserum to the appropriatecapsular polysaccharide--the encapsulated recombinants can be identifiedby the presence of an antigen-antibody precipitin "halo" surrounding thecolonies.

What is claimed as new and intended to be covered by Letters Patentis:
 1. A vaccine composition for the prevention of infectious diseasescomprising an immunogenically-effective amount of a geneticallyattenuated and stable avirulent bacterial strain derived from a virulentbacterial strain by introducing into said virulent strain by genetictransfer and recombination at least two mutations of the same phenotypewhich renders said virulent strain avirulent, while permitting saidavirulent strain to retain immunogenicity, together with an inertpharmacologically acceptable carrier.
 2. The vaccine composition ofclaim 1 wherein said phenotype is a temperature sensitive phenotypewhich suppresses growth at the normal body temperature of a host forsaid virulent strain.
 3. The vaccine composition of claim 2 wherein saidstrain contains three mutations of the same temperature-sensitivephenotype.
 4. The vaccine composition of claim 2 wherein saidtemperature-sensitive phenotype suppresses the replication of saidstrain at normal human body temperature.
 5. The vaccine composition ofclaim 1 wherein said phenotype suppresses the replication of said strainin the absence of a growth factor.
 6. The vaccine composition of claim 5wherein said growth factor is selected from the group consisting ofantibiotics, and nonhost metabolites.
 7. The vaccine composition ofclaim 6 wherein said growth factor is an antibiotic.
 8. The vaccinecomposition of claim 1 wherein said phenotype suppresses the formationof a toxic toxin from said strain.
 9. The vaccine composition of claim 1wherein said phenotype suppresses the invasiveness of said strain. 10.The vaccine composition of claim 1 wherein said bacterium is Haemophilusinfluenzae.
 11. The vaccine composition of claim 10 wherein said strainof H. influenzae contains at least two different mutations of the sametemperature-sensitive phenotype.
 12. The vaccine composition of claim 11wherein said ts phenotype suppresses the growth of said H. influenzaestrain at normal human body temperature.
 13. The vaccine composition ofclaim 10 wherein said bacterium is H. influenzae A214-A/3.
 14. Thevaccine composition of claim 10 wherein said bacterium is H. influenzaeA214-A/3-C2/13.
 15. The vaccine composition of claim 2 wherein saidphenotype is such that replication of said bacterium will continue forat most seven divisions after transfer to the non-permissivetemperature.
 16. The composition of claim 15 wherein the phenotype ofsaid bacterium is such that replication is allowed for three divisionsafter transfer to the non-permissive temperature.
 17. A method ofpreparing a bacterial vaccine which comprises:altering a virulentbacterial strain by introducing into said virulent strain at least twomutations of the same phenotype, thereby genetically attenuating saidstrain by rendering said virulent strain avirulent while allowing saidstrain to retain immunogenicity.
 18. The method of claim 17 wherein saidphenotype is a temperature sensitive phenotype which suppresses growthat the normal body temperature of a host for said virulent strain.
 19. Amethod of protecting an animal against infectious diseases whichcomprises inoculating said animal with a vaccine composition accordingto any of claims 1, 2, 5, 8 or
 9. 20. A method of protecting a humanagainst infection by H. influenzae which comprises inoculating saidhuman with a vaccine composition according to any of claims 10-12.